Targeted genome regulation and modification using transcription activator-like effectors.

REVIEW ARTICLE

Targeted genome regulation and modification using transcription activator-like effectors James N. F. Scott, Adam P. Kupinski and Joan Boyes School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, UK

Keywords chromatin markers; CRISPR/Cas9; genome engineering; TALE proteins; transcription regulation; zinc finger nucleases Correspondence J. Boyes, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK Tel: +44 (0)113 343 3147 E-mail: [email protected] (Received 5 June 2014, revised 7 August 2014, accepted 13 August 2014) doi:10.1111/febs.12973

Transcription activator-like effectors (TALEs) are immensely powerful new tools for genome engineering that can be directed to bind to almost any DNA sequence of choice. They originate from the Xanthomonas species of plant pathogenic bacteria and, in nature, these proteins increase the virulence of Xanthomonas. However, in 2009, the DNA binding code of TALEs was deciphered and, subsequently, TALE proteins have been exploited for many diverse applications. Custom TALEs that target almost any required DNA sequence can be readily constructed in < 1 week. One major application is gene editing: TALEs fused with the Fok I endonuclease catalytic domain can induce double-stranded breaks at a chosen genomic location, similar to zinc finger nucleases. Designer TALE transcription factors have also been developed by linking TALEs to a transcription AD, such as VP64. More recently, TALEs have been developed that can repress transcription, bind methylated DNA or act as fluorescent chromatin probes. In the present review, we describe the assembly of designer TALEs, their expanding range of current and potential future applications, and briefly discuss alternatives, namely, zinc finger nucleases and clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat associated protein 9.

Introduction The ability to specifically target effector proteins to any genomic DNA sequence of choice is a long-standing problem. Although some progress was made using zinc finger nucleases (ZFNs), recently, transcription activator-like effectors (TALEs) have been identified as being remarkably useful for this purpose. TALEs are naturally occurring DNA binding proteins found in the Xanthomonas spp. of plant pathogenic bacteria [1] with homologous proteins in other bacterial species, such as Ralstonia solanacearum [2,3]. In nature, these

type III effector proteins activate transcription of plant genes to give favourable growth conditions for the bacteria. For example, avrBs3, the first TALE that was isolated (from Xanthomonas campestris pv. vesicatoria), can induce a hypersensitive reaction in the ECW-30R strain of the pepper plant Capsicum annuum, resulting in necrotic leaf lesions. Following the discovery of the DNA binding property of TALEs in 2007, it was found that avrBs3 recognizes the pepper plant disease resistance gene Bs3, a gene that regulates

Abbreviations AD, activation domain; Cas9, CRISPR associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeats; cTALEN, compact TALEN; ESC, embryonic stem cell; FLASH, fast ligation-based automatable solid-phase high-throughput system; HDR, homology directed repair; LIC, ligation-independent cloning; NHEJ, nonhomologous end joining; NSC, neural stem cell; RTL, Ralstonia TALE-like protein; RVD, repeat variable diresidue; sgRNA, single-guide RNA; TALEN, TALE nuclease; TALE-TF, TALE transcription factor; TALE, transcription activator-like effector; TET1, ten–eleven translocation methylcytosine dioxygenase 1; TRE, tetracycline responsive element; ZFN, zinc finger nuclease.

FEBS Journal (2014) ª 2014 FEBS

1

TALEs – versatile DNA binding tools

cell size [4,5]. When avrBs3 binds to the promoter of this gene, the cells grow larger than normal, facilitating the growth of Xanothomonas [6]. Interestingly, the avrBs3 protein was found to contain 17.5 repeats of 34 amino acid residues that have 91–100% homology [7]. The intriguing repetitive nature of the DNA binding domain led to the binding code being deciphered in 2009; two back-to-back studies reported that residues 12 and 13 within the 34 amino acid repeat, form the repeat variable diresidue (RVD) and determine the nucleotide to which the particular module binds. Boch et al. [8] used a functional approach; they predicted the code by comparing the binding site and repeat domains of the TALE avrBs3 and confirmed this experimentally by using a b-glucuronidase reporter system. By comparison, Moscou and Bogdanove [9] used a bioinformatic approach by analyzing ten naturally occurring TALEs and their cognate DNA binding sequences. Both groups came to the same conclusion: an RVD of NN binds guanine and adenine with similar affinity, NG binds thymine, HD binds cytosine and NI binds adenine [8,9]. In addition to these four abundant repeats, another 19 naturally occurring RVDs were identified (Table 1). These include HI that binds adenine; ND that binds cytosine; and HN, NQ, NH and NK that each bind guanine, whereas the remaining RVDs show only very limited binding specificity [10]. Cracking this code enabled the production of designer TALEs that bind to any chosen DNA sequence and, by linking them to a range of effector domains, these proteins are proving to be immensely powerful tools in genome manipulation. The initial studies reported that the NN RVD is unable to discriminate between guanine and adenine [8,10]. However, other studies suggested that the NN RVD displays a preference for guanine [11–13] and the emerging consensus is that NN usually binds guanine but has a weak affinity for adenine, depending on the neighbouring RVDs. To overcome potential mis-targeting of TALEs via the NN motif, the alternative guanine-specific RVDs were characterized: NH [10,12] and NK [14] and were shown to function well in screens that used the TALEs as transcription activators (TALE transcription factor; TALE-TF) or as sequence specific nucleases (TALE nuclease; TALEN). Recently, several Ralstonia TALE-like proteins (RTLs) have been described [2,3]. These proteins share a structure very similar to TALE proteins and the RVDs present in RTLs have similar binding specificity, although they often show higher transcriptional activity compared to TALE RVDs [3]. Notably, the RTL RVDs retain their specific DNA binding activity when engineered into the Xanthomonas TALE protein 2

J. N. F. Scott et al.

Table 1. Binding specificities of the identified RVDs. RVD

A

T

G

C

NI HI HA NG HG NC HN NN NH NQ SS SN NK HD ND N* NA S* NV HH H* YG NS

++++ +++ + + ++ ++ + +++ ++ +++

++ ++ + – – – – – + ++ ++ –

+ + + + +++++ +++++ ++++ ++ + + + + +++ ++ +++

+ + + ++++++ ++ ++ +++ ++++ +

The RVDs that have been identified are listed together with their relative binding affinity for different nucleotides as determined by activation of a luciferase reporter gene [10]. The RVDs with the best increase in binding for a specific nucleotide over other nucleotides are shown in bold.

backbone, thus increasing the variety of available RVDs for TALE engineering [2,3]. Further analysis showed that TALE proteins consist of an N-terminal domain, which binds a thymine residue, a central DNA binding domain, comprised of a series of 34 amino acid repeat modules that each bind a single DNA base and differ only in their RVD sequence, a 0.5 repeat that contains an RVD, and a C-terminal domain (Fig. 1) [8]. To date, TALEs have been crystallized by two groups, who found that TALE proteins wrap around the DNA helix, with individual RVD modules binding within the major groove [15,16]. One of these crystal structures, where the TALE PthXo1 is bound to its DNA target, shows that an individual TALE repeat consists of two a-helices, connected by a loop, and it is this loop that contains the RVD that contacts the DNA [15]. Interestingly, the target sequence of all naturally occurring TALEs begins with a thymine at the very 50 end [8,9]; this is considered to be the result of a cryptic DNA binding signal within the N-terminal domain [17]. Indeed, a recently published crystal structure shows that the N-terminal domain features four continuous FEBS Journal (2014) ª 2014 FEBS



34-aa RVD modules A/G/C/T C T A C G T A C G T G T A

NH2

Effector domain

LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG RVD

NG = T

HD = C

NI = A

NH = G

Fig. 1. Schematic representation of a TALE protein. The four individual DNA binding modules are shown in red, green, yellow and blue; these bind the bases indicated. The amino acid sequence of a DNA binding module is shown, which is the same for all modules, apart from the residues highlighted in red, the RVD, which determines the base that the individual module binds.

repeats that contain two a-helices with a connecting loop: a fold similar to that of the main TALE DNA binding domain [18]. Notably, when the N-terminal domain is deleted, TALE/DNA binding is lost, whereas the N-terminal domain alone is capable of binding DNA, implying that this domain plays an essential role in DNA binding [18]. Significantly, one study showed that substitution of the 50 T in the target site of a TALE activator with C, G or A reduced the activity of the TALE by two- to three- fold [13] and another showed that the absence of a 50 T in the binding site could reduce activity by 10- to 1000-fold [19]. Although the N-terminal region does not have any sequence similarity to an RVD domain, recent experiments have allowed this 50 T binding constraint to be overcome. Two groups separately performed directed evolution experiments and showed that, by mutating the N–1 hairpin region of the N-terminal TALE domain, the affinity for alternative 50 bases is increased, effectively removing the 50 T binding constraint [19,20]. This therefore considerably simplifies TALE design and the choice of binding site. More recently, TALEs have been engineered to increase their DNA binding specificity by performing mutagenesis on the C-terminal part of the protein. Here, seven positively-charged arginine and lysine residues were hypothesized to nonspecifically bind to the negatively-charged DNA backbone. When these residues were replaced with glutamine, the resulting TALEN showed at least a four-fold higher specificity in an in vitro DNA cleavage assay [21]. In TALEs found in nature, the C-terminal domain carries an activation domain (AD) [1]. Importantly, this region can be replaced with other functional domains to generate TALEs that perform a range of other functions. For example, TALENs can be generated by replacing the AD with the catalytic domain of Fok I nuclease, or TALE-TFs can be formed by the addition of a new transcription AD, as described


below. Currently, the major applications of TALEs are gene editing (using TALENs) and gene activation (using TALE-TFs), although these uses are expanding significantly. In this review, both of these applications are discussed in detail and we expand this to describe the ever-increasing range of current applications, as well as future potential applications.

Production of TALEs Enthusiasm for the use of TALEs in genome engineering has been matched by an explosion in the methods used to generate them (Table 2). A major group of methods uses Golden Gate digestion–ligation, in which several different DNA fragments are ligated together in a specified order, via the use of a type II restriction enzyme that cleaves 1–5 bp away from its recognition site. This generates a series of unique sticky ends, allowing DNA fragments to be ligated only in the desired order. Upon digestion, the restriction site is removed, enabling simultaneous digestion/ligation in the same reaction mixture (Fig. 2). One such method of TALE assembly was described by Cermak et al. [22], in which TALE monomers (a TALE repeat unit containing one RVD) are contained within plasmids and are used directly in a Golden-Gate reaction. Multimers of up to 10 monomers can be assembled, followed by the ligation of three multimers, to produce TALEs that recognize up to 30 bp [22]. Sanjana et al. [17] described an alternative Golden Gate cloning method in which PCR is used to add unique restriction sites onto the ends of monomers. An advantage of this is that only four monomer plasmids are required, compared to the 60 plasmids used in the Cermak method. However, this suffers from the disadvantage that PCR may introduce mutations; nevertheless, TALEs that bind sequences of up to 24 bp can be routinely produced in ~ 1 week [17]. 3



Table 2. Overview of current methods for TALE production. Initial library size indicates the number of plasmids containing RVDs used in each of the methods. The maximum available length of TALE that can be produced in each method is indicated, including the last 0.5 RVD repeat. Method of production

Initial library size

Maximum TALE length

a

24.5

Golden Gate

50a

30.5

Golden Gate Golden Gate FLASH LIC

100 832a 372a 64a

Golden Gate/PCR

a

4

12.5 14.5 No limit 18.5

Assembly

Reference

Two-step assembly. First step: RVD monomers into hexamers. Second step: arrays into TALE. Both monomers and hexamers are PCR amplified Two-step assembly. First step: RVD monomers into arrays of 10. Second step: arrays into TALE One-step assembly into TALE One-step assembly into TALE Sequential ligation on a solid surface. Amenable to automation Two-step assembly. First step: dimers into arrays of six; second step: arrays of six into TALE. Amenable to automation

Sanjana et al. [17]

Cermak et al. [22] Li et al. [24] Ding et al. [23] Reyon et al. [25] Schmid-Burgk et al. [27]

Library available from Addgene (https://www.addgene.org/).

GG T C T C

G AG ACC

5’

3’

3’

5’ CC AG AG

BsaI recognition Cleavage site site

C T C T GG

Cleavage BsaI recognition site site Digestion of DNA fragments with BsaI generates unique sticky ends GGC A

ACT A

CG AC GC TG

TCAT

TGAT

+ Simultaneous digestion and ligation in the same reaction mixture ligates fragments in a specific order

Fig. 2. Golden Gate ligation. TALE monomers are flanked by sites for restriction enzymes, such as BsaI and BsmBI, which cleave 5 bp away from their recognition site. By flanking TALE modules with different DNA sequences within this 5 bp, a series of different sticky DNA ends can be generated, following digestion. This allows ligation of the TALE modules only in a specific order.

A third method based on Golden Gate ligation is a one step method developed by Ding et al. [23]. Here, a library of 832 plasmids, containing every 4-mer and 3mer combination of TALE repeat units is used. Plasmids containing the desired sequences are digested, and three 4-mers and one 3-mer are ligated directly into a plasmid that contains the TALE backbone to produce a TALE that recognizes a 15-bp target sequence [23]. The advantages of this method are that only a single ligation step is required and so it is faster 4

than the multiple step methods described above, and, by avoiding PCR, there is a lower risk of mutations. A similar approach has been described by Li et al. [24] where a library of 100 monomer and dimer RVDs allows one step assembly of up to 13 TALE repeats. High-throughput generation of TALEs High demand for TALEs led to the development of high-throughput methodology for their production. FEBS Journal (2014) ª 2014 FEBS


Fast ligation-based automatable solid-phase highthroughput system (FLASH) assembly uses a library of 376 pre-assembled TALE units (contained in plasmids), representing every monomer, dimer, trimer and tetramer combination [25]. Modules are ligated in order, depending on the target sequence of choice, to streptavidin coated magnetic beads, with unbound modules being washed off between each binding step. Release of the full-length TALE from the bead is then achieved by restriction enzyme digestion. Because this approach uses magnetic beads, it eliminates many intermediate steps including gel purification and PCR of intermediates used in the other Golden Gate methods described above [17]. Importantly, this allows TALE production to be automated: by using FLASH assembly and a liquid handling robot, 96 TALEs can be produced in < 1 day [25]. An alternate rapid, high-throughput method uses ligation-independent cloning (LIC) in which the exonuclease activity of T4 DNA polymerase ‘chews back’ the ends of the DNA fragments, resulting in fragments containing long nonpalindromic (10–30 nucleotides), single-stranded overhangs. Fragments with the appropriate complementary ends are then annealed and, given the length of the complementary region, do not dissociate upon transformation into Escherichia coli. These fragments are ligated by bacterial ligases and by including a fragment containing a bacterial replication origin in the assembly, the resulting plasmid can be propagated in Escherichia coli [26]. To assemble a TALE, a library of 64 dimers is used (each containing two RVDs) along with intermediate backbone fragments. First, three hexamers are produced, which are then ligated with an origin containing backbone and transformed. Because LIC is very efficient, the transformation does not need to be plated and, after overnight growth, it is used directly to prepare plasmid DNA. Following restriction digestion, these hexamers are used in a second round of LIC to produce a fulllength TALE. This LIC method has recently been adapted and automated, allowing over 600 functional TALEs to be produced in a single day [27]. Optimal TALE construction Naturally occurring TALEs are relative large proteins so to simplify their production and expression, several studies attempted to find a minimal region required for DNA binding activity [11,28,29]. A number of architectures for TALE design containing various N- and C-terminal deletions have been obtained. These commonly include the deletion of the most N-terminal 153 amino acids, which mediate transport of natural FEBS Journal (2014) ª 2014 FEBS


TALEs into plant cells and thus are dispensable for other functions [30], and various C-terminal truncations, where the natural transactivation domain is removed and further truncations are made to the C-terminus, but retaining 28–63 amino acids because these are required for TALE binding [11,28]. When constructing TALEs, the target DNA binding site needs to be carefully considered. Individual RVDs have been classified as ‘weak’, ‘intermediate’ and ‘strong’ [12]. Strong RVDs bind DNA with a high affinity via interactions that include hydrogen bonds, whereas weak RVDs use only van der Waals forces [12]. The strong RVDs include HD and NN, which bind to cytosine and guanine/adenine, respectively. However, NH, the RVD that binds to guanine more specifically than NN, binds to DNA with a lower affinity than NN and is classed as an intermediate RVD. Weak RVDs include NI and NG, which bind to adenine and thymine, respectively. For efficient TALE/ DNA binding, at least three or four strong RVDs must be present and stretches of weak RVDs must be avoided. Moreover, NN should only be used to recognize guanine if other strong RVDs are present to retain specificity [12]. These binding affinities therefore place some constraints on the sequence against which a TALE can be designed. An affinity bias for the 50 end of the TALE target sequence also has been reported [13,21]. In an in vitro target site selection assay, purified TALE proteins were presented with a library of DNA oligomers and the bound oligonucleotides were subjected to highthroughput sequencing [13]. In a second assay, the cleavage efficiency of purified TALEN proteins was assayed using a library of DNA oligonucleotides [21]. In both cases, increased tolerance for mismatches closer to the 30 end of the TALE target sequence was observed. To allow rapid and easy design of TALENs, several web-based tools are now available. These include TALENT [31], MOJO HAND [32], E-TALEN [33] and SAPTA [34]. They predict the optimal TALEN binding sites based on the length and base composition of the target site. In addition, the E-TALEN and SAPTA systems introduce a scoring system of potential target sites, based on experimental data, which allows TALEN activity at a chosen target to be predicted, thus improving specificity and reducing off target effects.

TALENs and gene editing TALEs are now used for a wide range of functions, as discussed further below, although the emergence of TALENs for genome editing was a major development.

5


Genome editing is achieved by linking TALEs to the catalytic domain of Fok I nuclease that catalyses the cleavage of one DNA strand. When two TALENs flank a section of DNA, the Fok I domains dimerize, allowing the catalysis of a double-strand DNA break to occur (Fig. 3) [11]. The distance between the two TALEN binding sites necessary for optimal DNA cleavage is determined by the size of the C-terminal domain retained on the TALE (i.e. the region that is then linked to Fok I nuclease). A 12–13 bp spacer is required when 28 amino acids of TALE are retained, whereas a longer spacer of 14–23 bp is preferred when the more commonly retained 63 amino acid domain is present, with maximal cutting efficiency occurring with a spacer between 14 and 19 bp [11]. TALEN-mediated gene editing can be achieved via two distinct mechanisms: (a) gene knockout by nonhomologous end joining (NHEJ) and (b) gene addition by homology directed repair (HDR) [35]. These have been successfully used in a number of organisms, including human somatic cells [11], human pluripotent cells [36], zebrafish [37–39], mouse [40], rat [41], tobacco [42], rice [43] and fruitfly [44]. The most common and simplest application of gene editing is to perform a gene knockout. TALENs are generated against the target gene, causing a doublestrand break. This is then repaired via the NHEJ machinery, which causes insertions or deletions into the gene of choice, leading to the generation of a frameshift mutation (Fig. 4A) [35]. This was used in human K562 cells to knock out the human CCR5 gene with modification efficiencies ranging from 5% to 27%, with > 15% gene modification observed with five TALEN pairs [11]. A major emerging use of TALENs is in the generation of knockout animals. Traditionally, this involved homologous recombination in embryonic stem cells (ESCs), blastocyst injection, followed by breeding of the heterozygous mice to generate homozygous null mice, with the entire process taking up to 1 year [45]. By contrast, genome editing technology using TALENs enables homozygous knockout mice to be obtained in < 2 months. This is because the desired gene can be directly targeted in fertilized eggs and the high efficiency of TALEN-mediated modification enables knockout of both alleles simultaneously [46]. Examples include the generation of Sry/ mice [40] and, notably, a recent study where three genes were knocked out simultaneously [47]: co-injection of mouse zygotes with TALEN pairs against the Agouti, miR205 and Arf genes successfully produced mice with mutations in all three genes, with a good efficiency (~ 7%) [47]. 6


The second major mode of gene editing by TALENs involves the addition of DNA sequences through HDR (Fig. 4B) [11,35]. This enables a DNA sequence to be inserted into the genome at a specific location and was first described using ZFNs, where insertions of up to ~ 8 kb have been successful, a feat also achieved more recently using TALENs [11,48]. TALENs are designed against the region where an insertion is required and are co-transfected into cells with a donor template that has ‘arms’ with sequence homology to the region on both sides of the double-stranded break. The doublestranded break is then repaired, using the donor DNA as a template, resulting in a precise integration of the donor sequence into the genome [48]. Notably, TALENs have been used to insert a BglI restriction site into the human CCR5 locus with up to 16% efficiency [11]. Once gene editing has been performed, cells with the desired mutation can be selected by isolating and growing single colonies and screening (e.g. by PCR) [23]. Importantly, TALENs or ZFNs (below) have huge potential in gene therapy because their use in HDR could overcome the safety concerns of current approaches whereby genes become randomly inserted into the genome. TALENs could also overcome the problem of chromatin-based effects on gene silencing because HDR enables the specific integration of DNA at a location of choice [48]. The clinical potential of this technology was highlighted very recently by the highly efficient TALE-mediated correction (76%) of the IL2RG locus, which, when mutated, leads to X-linked severe combined immunodeficiency [49]. Although TALENs have huge potential, they are not without their drawbacks. Toxicity can be an issue, although TALENs show less toxicity compared to ZFNs, with an average of 80% TALEN expressing cells surviving for 5 days post-transfection compared to 50% of ZFNs expressing cells [50]. One key cause of toxicity is if a TALEN pair is not sufficiently specific, which can result in nonspecific cutting at different points in the genome, leading to genomic instability and cell death through the knockdown of vital genes. Indeed, although RVDs preferentially bind to specific nucleotides, they also have some binding capacity for other nucleotides (Table 1), which can contribute to these off target effects. Also, the most commonly used endonuclease, Fok I, functions as a homodimer but, if two identical TALENs interact rather than two TALENs flanking the desired target site, this can lead to increased off-target DNA cutting. To reduce such toxic effects, several variants of heterodimeric Fok I have been developed [51–53]. Recently, a single-chain TALEN architecture has been developed to simplify the production and delivery FEBS Journal (2014) ª 2014 FEBS



14–20 bp TALEN A N

C

DSB

FokI FokI

C

N TALEN B

Fig. 3. TALEN-mediated DNA cleavage. TALEN A and TALEN B are designed so that they bind opposite strands of DNA and are 14–20 bp apart. This allows the Fok I domains that are attached to the C-terminal domain of each TALEN to dimerize and catalyse a double-strand break (DSB) between the two TALE binding sites.

of TALENs to target cells. These compact TALENs (cTALENs) contain the cleavage domain from I-TevI homing endonuclease fused to the TALE DNA binding domain. By contrast to the standard TALEN architecture, the cleavage domain of cTALEN is fused at the N-terminus to achieve double-strand breaks. Interestingly, the activity of I-TevI domain located at the C-terminus results in DNA nicks rather than doublestrand breaks. Nicking preferentially induces homologydirected repair and shows a 25-fold increase in targeted gene conversion over the standard Fok I-based TALEN [54].

TALE-TFs The ability to pinpoint specific sites in the genome with high accuracy means that TALEs can be used to modulate the expression of individual genes by linking an activation or repression domain to TALEs. A commonly used AD is VP64, the minimal AD of the herpes simplex virus protein VP16, which efficiently

activates transcription when bound close to a promoter in mammalian cells [55]. Activation by TALE-TFs was comprehensively examined by Zhang et al. [28]. Specifically, TALEs linked to a VP64 AD were used to activate mCherry reporter constructs that carried binding sites for their respective TALE. Transfection of the reporter construct alone (without a TALE-TF) resulted in no fluorescence. However, co-transfection of a TALE-TF resulted in activation of the reporter by all of the TALEs tested (targeted against a range of different binding sites), with all but one generating more than five-fold induction. Endogenous genes were also induced; TALEs generated against the promoter of SOX2 and KLF4 upregulated gene expression by 5.5 0.1-fold and 2.2 0.1-fold, respectively. However, TALE-TFs generated against c-MYC and OCT4 promoters did not change their expression, possibly as a result of these genes being in repressed chromatin in the cell type used (293FT cells), as discussed below [28].

TALEN induced double strand break

A NHEJ mediated repair

B HDR mediated repair

Deletion mutations: + Donor template Insertion mutations:

Precise nucleotide alterations or sequence insertion

Fig. 4. Mechanisms by which TALENs can be used for gene editing. (A) NHEJ-mediated repair: DNA break repair by the nonhomologous end joining pathway is error prone and can lead to the generation of frameshift mutations through insertion (green) or deletion (red) of nucleotides. (B) HDR-mediated repair: break repair by homology-directed repair leads to precise alterations/insertions from a double-stranded DNA donor template (green).


7


Linking TALEs to the VP64 transcription domain gives a very high level of expression, and this is not always desirable [55]. Instead, TALEs can be linked to a weaker transcription AD and, to this end, the p65 AD of NF-jB, has been used. For example, TALEs linked to p65 were able to effectively induce expression of an AmCyan reporter gene but at three-fold lower levels than VP16 fusions [56]. Following the same principle by which TALEs activate transcription, TALEs can be used as transcriptional repressors by linking them to either SID (mSin interaction domain from Mad [57]) or to KRAB (Kr€ uppel-associated boxes, DNA binding-dependent transcriptional repressors [58]) domains. TALEs linked to these domains have successfully orchestrated the repression of endogenous mammalian genes; for example, SOX2 transcription was decreased by three-fold in HEK 293FT cells using a TALE with a SID repressor and more than two-fold using a TALE with a KRAB repressor [10]. Recently, TALE activators (and repressors) were found to regulate gene expression in a position- and strand-dependent manner in mammalian cells [59]. TALE activators robustly activated the transcription of a reporter gene when they were targeted to a site within the promoter on either the sense or antisense strand, and also activated transcription when the binding site was located on the antisense strand downstream of the promoter (in the 50 UTR). Interestingly, location of the binding site on the sense strand of the 50 UTR inhibited transcription; moreover, this inhibitory effect was also observed when the TALE effector domain was removed, suggesting that the TALE may block the passage of RNA polymerase II [59]. These results therefore imply that the TALE binding site needs to be carefully selected to achieve optimal gene activation or repression.

Other applications TALEs have huge potential beyond genome editing and gene regulation. Recent examples include the use of TALEs as chromatin modifiers, demethylases and as fluorescent chromatin probes. Further potential uses have been proposed; for example, as antivirals by targeting viral genomes with TALENs [60]. Key examples of these emerging uses are summarized below. TALE switch and two-hybrid system In an extension of the above gene transcription studies, TALEs have been used to switch on and then later switch off gene expression [56]. A stable HEK293 cell 8


line was constructed with a synthetic fluorescent reporter gene, AmCyan, under the control of a tetracycline responsive element (TRE) and a minimal cytomegalovirus promoter. Activating TALE-TFs (in frame with the VP16 transactivation domain) that bind the TRE was shown to strongly activate the expression of AmCyan. Interestingly, transfection of 10 ng of one particular TALE-VP16 fusion against this region was able to induce AmCyan expression to a higher level than a saturation concentration of doxycycline (1 lgmL1). Repressive TALEs (in frame with the KRAB repressive domain), which also bind the TRE, could then abolish this activation. Indeed, transfection of 150 ng of a TALE-KRAB fusion completely abolished the expression of AmCyan activated by 10 ng of the activation TALE [56]. These studies were further developed to generate an inducible TALE-based two-hybrid system that allows temporal modulation of gene expression. Here, a TALE against the TRE of AmCyan was fused to the Rheo Receptor (New England Biolabs, Beverly, MA, USA). This was co-transfected with the Rheo activator, which has a VP16 AD, although this could not activate expression of the target genes alone. Upon induction with GenoStat ligand (Millipore, Billerica, MA, USA), the Rheo activator and receptor form a heterodimer. This brings the VP16 domain into the proximity of the TALE-bound target gene, allowing induction of gene expression [56]. This system therefore allows the activity of a TALE to be tightly regulated, depending on the concentration of GenoStat ligand added and means that different levels of gene induction can be attained, as well as enabling the induction of gene expression on specific time scales [56]. Fluorescent TALE probes A significant recent development is the ability to use TALEs as fluorescent probes to monitor chromatin dynamics. In proof of principle experiments, Miyanari et al. [61] generated TALE-fluorescent protein fusions against repetitive (minor satellite and major satellite) regions of the mouse genome. This allowed the nuclear positions of these target sequences to be visualized in real time by confocal microscopy [61]. Ma et al. [62] developed similar TALE-fluorescent protein fusions but, in addition, showed that the signal from TALEfluorescent proteins targeted to telomeres positively correlated with telomere length, and that populations with differing telomere length could be detected by flow cytometry. Currently, fluorescent TALE probes can only be used to monitor the dynamics of repetitive FEBS Journal (2014) ª 2014 FEBS


regions of the genome because the signal to noise ratio precludes detection of a single copy locus. Indeed, it is likely that multiple TALEs against a single copy locus will be required [61]. However, once this hurdle is overcome, the potential to monitor the dynamics of almost any region of the genome will be immensely powerful.

TALE binding to chromatin The ability of TALEs to function critically relies on their ability to bind to their target sites in chromatin. Notably, not all TALEs constructed against a particular region are active; sometimes, several TALEs that bind to a single gene must be produced, of which only one may have the required activity, and in some cases, all TALEs produced against a particular region may be inactive. For example, as outlined above, Zhang et al. [28] showed that SOX2 and KLF4 are successfully activated by TALE-TFs in 293FT cells but TALE-TFs that target the c-MYC and OCT4 promoters did not alter their expression [28]. This was considered to be a result of these genes being inactive in the cell type used. Similarly, for gene editing, several pairs of TALENs are usually constructed against the gene of interest because not all TALENs are active [17]. In an attempt to improve TALE efficiency, the ability of TALEs to synergize was investigated. Importantly, the binding of more than one TALE activator was indeed found to have a stronger effect than the additive effect of individual TALEs [63,64]. This was demonstrated using TALEs with either a VP64 or a p65 effector domain and when activating both a coding gene (VEGF-A) and a noncoding miRNA gene cluster (mIR-302/367) [63]. In a related study in our laboratory, we systematically assessed the binding of a TALE to chromatin substrates and found that, in vitro, the TALE can bind to its target site when this is close to the entry and exit sites of a nucleosome but not when the binding site is at the nucleosome dyad. Furthermore, we showed that, in vivo, TALEs can bind to transcriptionally repressed chromatin and that transcription through the TALE binding site increases binding by only approximately two-fold [65]. Our results therefore suggest that TALEs can bind to repressed chromatin in at least some cells of the population. However, the inability of TALEs to bind when their binding site falls at the nucleosome dyad implies that TALEs are unlikely to bind their target in all cells of the population. Furthermore, occlusion of the TALE binding site by a positioned nucleosome could explain why some TALEs fail to function. Together, these experiments reinforce the idea that using multiple FEBS Journal (2014) ª 2014 FEBS


TALEs with adjacent binding sites will increase the chances of successful TALE binding and an effect in vivo [26]. TALE binding to methylated DNA Methylation of CpG dinucleotides is the major modification of vertebrate DNA, with ~ 70% of all CpG dinucleotides being methylated. However, numerous studies suggested that DNA methylation impedes TALE function and, notably, the RVD that binds cytosine (HD) does not recognize methyl-cytosine [66,67]. This was seen as a limitation in the use of TALEs because promoter hypermethylation correlates with silencing of a significant fraction of genes [66]. Initial studies to achieve TALE-mediated activation of methylated promoters relied on treatment with 5-aza2-deoxycytidine, a suicide inhibitor of the maintenance methyltransferase, DNMT1 [68]. For example, one study found that TALEs activate OCT4 expression in ESCs but not in ESC-derived neural stem cells (NSCs). By using a combination of 5-aza-20 -deoxycytidine and valproic acid, inhibitors of DNA methyltransferases and histone deacetylases, respectively, TALE-TFs were able to activate OCT4 in NSCs at 60% of the endogenous level in ESCs. Notably, activation of OCT4 in NSCs in this way also resulted in the activation of OCT4 target genes, raising the intriguing possibility that TALEs could provide an alternative means of reprogramming somatic cells to pluripotency [68]. A subsequent and major step forward in the ability to activate methylated promoters was the construction of TALEs that can bind to 5-methylcytosine. To date, two different RVDs that recognize methylated cytosine have been published. Deng et al. [66] described the use of the RVD NG, which normally binds thymine, and Valton et al. [67] described the use of the RVD N*, where the second residue of the RVD is deleted. Valton et al. [67] propose that N* is preferable because it recognizes methyl cytosine better than NG. However, neither RVD exclusively binds methylated cytosine, meaning that the overall specificity of the TALE is reduced. Despite specificity limitations, these newly discovered RVDs greatly expand the efficacy of TALEs. More recently, TALEs have been fused to the ten– eleven translocation 1 (TET1) hydroxylase catalytic domain, which catalyzes the first step in the active demethylation of 5-methylcytosine, the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. These TALE-TET1 fusions selectively demethylated and activated expression of KLF4, RHOXF2 and HBB in three different human cell lines. Demethylation was mostly

9


observed within 30 bp of either end of the TALE binding site, although, in some cases, it was observed 150–200 bp away, suggesting that different chromatin states may play a role in the efficacy of these proteins. The efficiency of the TALE-TET1 fusions was variable, with up to 84% CpG demethylation at the HBB locus but only 25% CpG demethylation at the RHOXF2 locus in HEK293 cells. This could be a result of locus-dependent effects; for example, the level of DNA methylation, the ability of the TALE to bind to its target site or the accessibility of the TET1 catalytic domain. Nevertheless, these TALE-TET1 fusions are likely to have many applications both in research, with respect to studying the effects of DNA demethylation at specific sites, and in therapeutics, for example by reactivating repressed genes (as an alternative to TALE-TFs) [69]. Together, these different strategies mean that it should now be possible to target TALEs to most sites in the genome, thereby expanding their power and utility.

TALE alternatives in genome manipulation: ZFNs and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 (Cas9)


15 years and, in one case, ZFNs are being tested in a human phase II clinical trial [76,77]. This raises the question of why should TALENs be utilized if ZFNs are already a tried and tested method of gene editing? The main reasons for choosing TALENs over ZFNs are time and the cost of production, as well as the mechanism by which these proteins bind to DNA. For TALEs, each module binds one individual nucleotide, whereas, with ZFNs, each zinc finger binds 3 bp of DNA. Because zinc fingers that bind all possible 3-bp combinations are not available, a narrower range of DNA sequences can be specifically targeted by ZFNs than with TALENs. ZFNs are also subject to closely guarded intellectual property, with key patents for the design of ZFNs and for their use in drug discovery and gene regulation being held by Sangamo Biosciences (Richmond, CA, USA). This poses financial and technical barriers (as a result of license agreements) to using ZFNs in research [78]. TALEs, on the other hand, are an open technology, and genes have successfully been targeted using TALEs in a number of different organisms, including yeast [22], plants [22,79], zebrafish [39,80] and human cells [28]. As a result of TALEs being simpler to construct technically, a TALE targeting nearly any DNA sequence can be constructed using a kit for < $350 [76].

ZFNs Before the discovery of TALEs, designer DNA binding proteins could only be constructed using zinc finger proteins [70–72]. Zinc fingers form the DNA binding domain in a range of transcription factors, including Sp1, TFIIIA and Kr€ uppel [73]. Crystallography studies showed that the Cys2-His2 zinc finger proteins contain a a-helix that fits directly into the major groove of DNA, with the side chains of residues in the amino terminus of this helix forming contacts with DNA. Notably, each zinc finger contacts 3 bp of DNA, suggesting that different DNA sequences could be targeted by constructing proteins containing a series of different zinc finger modules [73,74]. The main application of these designer proteins is gene editing through the production of ZFNs. ZFNs consist of a series of zinc finger DNA binding modules, linked to the catalytic domain of Fok I. In addition, custom transcription factors have been generated by linking a zinc finger DNA binding domains to a transactivation domain (e.g. VP64). Importantly, all of the applications for zinc finger proteins have been recapitulated with TALEs [28,55,75]. TALENs are a relatively new technology, whereas ZFN technology has been under development for 10

CRISPR/Cas9 More recently, CRISPR/Cas9 technology has emerged as a highly effective alternative to TALENs and ZFNs in genome editing [81,82]. In nature, CRISPR/ Cas systems provide acquired immunity in bacteria against foreign DNA, by using a short RNA to direct the degradation of foreign nucleic acids. This system has been exploited by using the Cas9 nuclease from the well-characterized type II CRISPR system of Streptococcus pyogenes. Cas9 is guided to a target site by a single-guide RNA (sgRNA), containing a 20-bp target sequence that binds to the target DNA by Watson–Crick base pairing (Fig. 5) [82]. The target sequence must immediately precede a 50 -NGG protospacer adjacent motif, which imposes a small limitation on the exact location of the target site in the genome [82]. However, unlike TALENs and ZFNs, Cas9 cleaves at a precise location, 3 bp upstream of the protospacer adjacent motif sequence [83]. One significant advantage of CRISPR/Cas9 technology over TALENs and ZFNs is that they can be retargeted to a different site much more easily by using an alternative sgRNA that matches a different 20-bp target sequence [82]. FEBS Journal (2014) ª 2014 FEBS



So far, genome editing has been the main application of the CRISPR/Cas9 system because cleavage is the natural function of the Cas9 endonuclease. However, by using a nuclease deficient mutant of Cas9, alternative effector domains can be fused to Cas9, similar to the fusion of different effector domains to TALEs. For example, a nuclease-deficient Cas9-VP64 fusion has been demonstrated to activate target genes; however, the efficiency varied significantly, which could be a result of the chromatin environment of the target sequence [81]. Because Cas9 recognition relies on base pairing of the sgRNA with the target DNA sequence, it is possible that the entire target sequence must be accessible within chromatin to allow both melting of the template, and access of the sgRNA. This may differ from TALEs where we speculate that binding of one or more RVD domains could facilitate ‘peeling back’ of DNA on the nucleosome to allow further domains to bind. Even though there is the potential of using CRISPR/Cas9 systems for a range of different applications, it remains to be seen whether these applications will be as diverse as those involving TALEs. Notably, CRISPR/Cas9 is very effective in the production of knockout mice. As explained above, traditional methods are very time consuming, although the CRISPR based system enables knockout mice to be generated in just one step [84]. The components are injected directly into the zygote: for gene knockout, Cas9 and a guide RNA are injected and, for more precise alterations or conditional knockouts, an oligonucleotide with the desired modification is also injected (for integration by HDR). Using this technology, epitope tags, loxP sites and even fluorescent proteins have been added to endogenous genes. Importantly, off target effects were low: these were only observed at sites with one or two mismatches and so they could be abrogated by careful target site selection. Because the components are injected directly into the zygote, this system could be directly adapted to other mammalian species, including those where ES cells are not available [84]. A notable example is the first targeted gene

knockouts in monkeys of Ppar-c and Rag1, which had not been achieved previously using any other system [85]. Very recently, similar to the TALE system described above, a CRISPR/Cas9 system for monitoring chromatin dynamics has been described [86]. However, this derivative excels over the TALE version by having the capability to image single genomic loci. This is possible because of the fundamental targeting mechanism of the CRISPR/Cas9 system: multiple positions can be targeted within a single gene by introducing only a single Cas9-fluorescent protein fusion, together with a series of sgRNAs. This is unlike TALEs, where multiple constructs expressing a series of TALE-fluorescent protein fusions would be required. By introducing a single Cas9 fusion into cells, with a range of 16–73 sgRNAs, Chen et al. [86] reported the effective visualization of a nonrepetitive region of a single gene, MUC4. The ability to monitor chromatin dynamics with single loci resolution in live cells is likely to comprise a very powerful technique [86].

Conclusions The use of TALEs as a tool in research is still very much in its infancy, although it is already apparent that they are extremely powerful. TALEs are becoming a very important tool for many different applications in molecular biology, especially for genome editing. In addition, the development of vast range of other applications, including linking TALEs to fluorescent proteins to investigate genome dynamics or to DNA modifiers, such as TET1 to demethylate and activate genes, demonstrates that the uses of TALE technology are diverse. Even though a wide range of applications has already been developed, there is considerable potential for further new applications for these genome-targeting tools. For example, TALEs and Cas9 can be linked to chromatin modifiers to target epigenetic marks to specific regions of the genome, thus allowing the effects of these modifications at specific sites to be analyzed. In

Cas9 5’ 3’

Cleavage

sgRNA NGG NCC

3’ 5’

Fig. 5. CRISPR/Cas9 mediated cleavage. sgRNA (red line) binds to the target sequence by Watson–Crick base pairing. Cleavage, catalysed by Cas9, occurs 3 bp upstream of the NGG protospacer adjacent motif (PAM). The red arrow indicates the cleavage site.


11


addition, TALEs could be used as a probe for chromatin immunoprecipitation, followed by MS to determine which factors are bound to a particular region of the genome. Furthermore, an exciting new possibility is to use TALEs or CRISPR/Cas9 clinically to correct genes mutated in disease once the appropriate delivery vehicles have been developed. One of the major advantages of using TALEs is that they can perform functions similar to those of zinc finger proteins, with the potential for higher specificity, and without the barriers, especially for research in academia, with respect to the patents surrounding ZFN technology. It will be interesting to determine whether the emergence of CRISPR/Cas9 technology will succeed TALEs or complement their use in future applications. Nevertheless, the development of new applications of ZFN, TALE and CRISPR/Cas9 technology will continue to represent a very exciting field of research over the next few years.

Acknowledgements This work was funded by a Lady Tata Memorial Trust studentship (to JNFS), Yorkshire Cancer Research and a start-up grant from Children with Cancer (to APK).

Author contributions JNFS, APK and JB all contributed to the writing of the paper.

References 1 Boch J & Bonas U (2010) Xanthomonas AvrBs3 familytype III effectors: discovery and function. Annu Rev Phytopathol 48, 419–436. 2 Li L, Atef A, Piatek A, Ali Z, Piatek M, Aouida M, Sharakuu A, Mahjoub A, Wang G, Khan S et al. (2013) Characterization and DNA-binding specificities of Ralstonia TAL-like effectors. Mol Plant 6, 1318–1330. 3 De Lange O, Schreiber T, Schandry N, Radeck J, Braun KH, Koszinowski J, Heuer H, Strauß A & Lahaye T (2013) Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease. New Phytol 199, 773–786. 4 R€ omer P, Hahn S, Jordan T, Strauss T, Bonas U & Lahaye T (2007) Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science 318, 645–648. 5 Kay S, Hahn S, Marois E, Hause G & Bonas U (2007) A bacterial effector acts as a plant transcription factor and induces a cell size regulator. Science 318, 648–651.

12


6 Pennisi E (2012) The tale of the TALEs. Science 338, 1408–1411. 7 Bonas U, Stall RE & Staskawicz B (1989) Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol Gen Genet 218, 127–136. 8 Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A & Bonas U (2009) Breaking the code of DNA binding specificity of TALtype III effectors. Science 326, 1509–1512. 9 Moscou MJ & Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501. 10 Cong L, Zhou R, Kuo Y, Cunniff M & Zhang F (2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun 3, 968. 11 Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ et al. (2010) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29, 143–148. 12 Streubel J, Bl€ ucher C, Landgraf A & Boch J (2012) TAL effector RVD specificities and efficiencies. Nat Biotechnol 30, 593–595. 13 Meckler JF, Bhakta MS, Kim M-S, Ovadia R, Habrian CH, Zykovich A, Yu A, Lockwood SH, Morbitzer R, Els€aesser J et al. (2013) Quantitative analysis of TALEDNA interactions suggests polarity effects. Nucleic Acids Res 41, 4118–4128. 14 Christian ML, Demorest ZL, Starker CG, Osborn MJ, Nyquist MD, Zhang Y, Carlson DF, Bradley P, Bogdanove AJ & Voytas DF (2012) Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues. PLoS ONE 7, e45383. 15 Mak AN, Bradley P, Cernadas RA, Bogdanove AJ & Stoddard BL (2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335, 716–719. 16 Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu J-K, Shi Y & Yan N (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335, 720–723. 17 Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G & Zhang F (2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7, 171–192. 18 Gao H, Wu X, Chai J & Han Z (2012) Crystal structure of a TALE protein reveals an extended N-terminal DNA binding region. Cell Res 22, 1716–1720. 19 Lamb BM, Mercer AC & Barbas CF (2013) Directed evolution of the TALE N-terminal domain for recognition of all 50 bases. Nucleic Acids Res 41, 9779–9785.



20 Tsuji S, Futaki S & Imanishi M (2013) Creating a TALE protein with unbiased 50 -T binding. Biochem Biophys Res Commun 441, 262–265. 21 Guilinger JP, Pattanayak V, Reyon D, Tsai SQ, Sander JD, Joung JK & Liu DR (2014) Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods 11, 429–435. 22 Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ & Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39, e82. 23 Ding Q, Lee Y-K, Schaefer EA, Peters DT, Veres A, Kim K, Kuperwasser N, Motola DL, Meissner TB, Hendriks WT et al. (2013) A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 12, 238–251. 24 Li L, Piatek MJ, Atef A, Piatek A, Wibowo A, Fang X, Sabir JSM, Zhu J-K & Mahfouz MM (2012) Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification. Plant Mol Biol 78, 407–416. 25 Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD & Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30, 460–465. 26 Schmid-Burgk JL, Xie Z, Frank S, Virreira Winter S, Mitschka S, Kolanus W, Murray A & Benenson Y (2012) Rapid hierarchical assembly of medium-size DNA cassettes. Nucleic Acids Res 40, e92. 27 Schmid-Burgk JL, Schmidt T, Kaiser V, H€ oning K & Hornung V (2013) A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nat Biotechnol 31, 76–81. 28 Zhang F, Cong L, Lodato S, Kosuri S, Church GM & Arlotta P (2011) Efficient construction of sequencespecific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29, 149–153. 29 Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, Kim S, Lee C, Jeong E, Chung E et al. (2013) A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31, 251–258. 30 Szurek B, Rossier O, Hause G & Bonas U (2002) Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell. Mol Microbiol 46, 13–23. 31 Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, Vandyk JK & Bogdanove AJ (2012) TAL Effector-nucleotide targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res 40, W117–W122. 32 Neff KL, Argue DP, Ma AC, Lee HB, Clark KJ & Ekker SC (2013) Mojo Hand, a TALEN design tool for genome editing applications. BMC Bioinformatics 14, 1.



33 Heigwer F, Kerr G, Walther N, Glaeser K, Pelz O, Breinig M & Boutros M (2013) E-TALEN: a web tool to design TALENs for genome engineering. Nucleic Acids Res 41, e190. 34 Lin Y, Fine EJ, Zheng Z, Antico CJ, Voit RA, Porteus MH, Cradick TJ & Bao G (2014) SAPTA: a new design tool for improving TALE nuclease activity. Nucleic Acids Res 42, e47. 35 Joung JK & Sander JD (2012) TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14, 49–55. 36 Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC et al. (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29, 731–734. 37 Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK & Yeh J-RJ (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29, 697–698. 38 Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG II, Tan W, Penheiter SG, Ma AC, Leung AYH et al. (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491, 114–118. 39 Huang P, Xiao A, Zhou M, Zhu Z, Lin S & Zhang B (2011) Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29, 699–700. 40 Kato T, Miyata K, Sonobe M, Yamashita S, Tamano M, Miura K, Kanai Y, Miyamoto S, Sakuma T & Yamamoto T et al. (2013) Production of Sry knockout mouse using TALEN via oocyte injection. Sci Rep 3, 3136. 41 Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L et al. (2011) Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 29, 695–696. 42 Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ & Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161, 20–27. 43 Li T, Liu B, Chen CY & Yang B (2014) TALEN utilization in rice genome modifications. Methods, pii: S1046-2023(14)00121-2. doi: 10.1016/j.ymeth.2014. 03.019. [Epub ahead of print] 44 Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y, Zhang B et al. (2012) Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. J Genet Genomics 39, 209–215. 45 Hall B, Limaye A & Kulkarni AB (2009) Overview: generation of gene knockout mice. Curr Protoc Cell Biol, Chapter 19, Unit 19.12 19.12.1–17. 46 Aida T, Imahashi R & Tanaka K (2014) Translating human genetics into mouse: the impact of ultra-rapid in vivo genome editing. Dev Growth Differ 56, 34–45.

13


47 Li C, Qi R, Singleterry R, Hyle J, Balch A, Li X, Sublett J, Berns H, Valentine M, Valentine V et al. (2014) Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes. Mol Cell Biol 34, 1649–1658. 48 Moehle EA, Moehle EA, Rock JM, Rock JM, Lee YL, Lee YL, Jouvenot Y, Jouvenot Y, DeKelver RC, Dekelver RC et al. (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci USA 104, 3055–3060. 49 Matsubara Y, Chiba T, Kashimada K, Morio T, Takada S, Mizutani S & Asahara H (2014) Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus. Sci Rep 4, 5043. 50 Mussolino C, Alzubi J, Fine EJ, Morbitzer R, Cradick TJ, Lahaye T, Bao G & Cathomen T (2014) TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res 42, 6762–6773. 51 Doyon Y, Vo TD, Mendel MC, Greenberg SG, Wang J, Xia DF, Miller JC, Urnov FD, Gregory PD & Holmes MC (2011) Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 8, 74–79. 52 Guo J, Gaj T & Barbas CF (2010) Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J Mol Biol 400, 96–107. 53 Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA et al. (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25, 778–785. 54 Beurdeley M, Bietz F, Li J, Thomas S, Stoddard T, Juillerat A, Zhang F, Voytas DF, Duchateau P & Silva GH (2013) Compact designer TALENs for efficient genome engineering. Nat Commun 4, 1762. 55 Sadowski I, Ma J, Triezenberg S & Ptashne M (1988) GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563–564. 56 Li Y, Moore R, Guinn M & Bleris L (2012) Transcription activator-like effector hybrids for conditional control and rewiring of chromosomal transgene expression. Sci Rep 2, 897. 57 Ayer DE, Laherty CD, Lawrence QA, Armstrong AP & Eisenman RN (1996) Mad proteins contain a dominant transcription repression domain. Mol Cell Biol 16, 5772–5781. 58 Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ & Rauscher FJ (1994) Kr€ uppel-associated boxes are potent transcriptional repression domains. Proc Natl Acad Sci USA 91, 4509–4513.

14


59 Uhde-Stone C, Cheung E & Lu B (2014) TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells. Biochem Biophys Res Commun 443, 1189–1194. 60 Chen J, Zhang W, Lin J, Wang F, Wu M, Chen C, Zheng Y, Peng X, Li J & Yuan Z (2014) An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases. Mol Ther 22, 303–311. 61 Miyanari Y, Ziegler-Birling C & Torres-Padilla M-E (2013) Live visualization of chromatin dynamics with fluorescent TALEs. Nat Struct Mol Biol 20, 1321–1324. 62 Ma H, Reyes-Gutierrez P & Pederson T (2013) Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors. Proc Natl Acad Sci USA 110, 21048–21053. 63 Maeder ML, Linder SJ, Reyon D, Angstman JF, Fu Y, Sander JD & Joung JK (2013) Robust, synergistic regulation of human gene expression using TALE activators. Nat Methods 10, 243–245. 64 Perez-Pinera P, Ousterout DG, Brunger JM, Farin AM, Glass KA, Guilak F, Crawford GE, Hartemink AJ & Gersbach CA (2013) Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat Methods 10, 239–242. 65 Scott JNF, Kupinski AP, Kirkham CM, Tuma R & Boyes J (2014) TALE proteins bind to both active and inactive chromatin. Biochem J 158, 153–158. 66 Deng D, Yin P, Yan C, Pan X, Gong X, Qi S, Xie T, Mahfouz M, Zhu J-K, Yan N et al. (2012) Recognition of methylated DNA by TAL effectors. Cell Res 22, 1502–1504. 67 Valton J, Dupuy A, Daboussi F, Thomas S, Marechal A, Macmaster R, Melliand K, Juillerat A & Duchateau P (2012) Overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation. J Biol Chem 287, 38427–38432. 68 Bultmann S, Morbitzer R, Schmidt CS, Thanisch K, Spada F, Elsaesser J, Lahaye T & Leonhardt H (2012) Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers. Nucleic Acids Res 40, 5368–5377. 69 Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, Ho QH, Sander JD, Reyon D, Bernstein BE et al. (2013) Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol 31, 1137–1142. 70 Bibikova M, Golic M, Golic KG & Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175.



71 Bibikova M, Beumer K, Trautman JK & Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764. 72 Urnov FD, Rebar EJ, Holmes MC, Zhang HS & Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636–646. 73 Pavletich NP & Pabo CO (1991) Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809–817. 74 Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188, 773–782. 75 Gommans WM, Haisma HJ & Rots MG (2005) Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command. J Mol Biol 354, 507–519. 76 DeFrancesco L (2011) Move over ZFNs. Nat Biotechnol 29, 681–684. 77 Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, Spratt SK, Surosky RT, Giedlin MA, Nichol G et al. (2014) Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 370, 901–910. 78 Scott CT (2005) The zinc finger nuclease monopoly. Nat Biotechnol 23, 915–918. 79 Mahfouz MM, Li L, Shamimuzzaman M, Wibowo A, Fang X & Zhu J-K (2011) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 108, 2623–2628.



80 Moore FE, Reyon D, Sander JD, Martinez SA, Blackburn JS, Khayter C, Ramirez CL, Joung JK & Langenau DM (2012) Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs). PLoS One 7, e37877. 81 Mali P, Esvelt KM & Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10, 957–963. 82 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA & Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281–2308. 83 Xu T, Li Y, Van Nostrand JD, He Z & Zhou J (2014) Cas9-based tools for targeted genome editing and transcriptional control. Appl Environ Microbiol 80, 1544–1552. 84 Yang H, Wang H, Shivalila CS, Cheng AW, Shi L & Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/ Cas-mediated genome engineering. Cell 154, 1370– 1379. 85 Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W et al. (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNAmediated gene targeting in one-cell embryos. Cell 156, 836–843. 86 Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li G-W, Park J, Blackburn EH, Weissman JS, Qi LS et al. (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491.

15

Cas9 System.

Targeted genome modification via triple helix formation.

Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

[Targeted genome modifications using TALEN].

Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology.

Genome-Wide Epigenetic Regulation of Gene Transcription in Maize Seeds.

Transcription factors as readers and effectors of DNA methylation.

Corrigendum: Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases.

sgRNA.

Transcription activator-like effectors: a toolkit for synthetic biology.

Phosphatase activities in spinach thylakoid membranes-effectors, regulation and location.

Draft genome sequence of Xanthomonas axonopodis pv. glycines 8ra possessing transcription activator-like effectors used for genetic engineering.

Regulation of transcription by the Arabidopsis UVR8 photoreceptor involves a specific histone modification.

Genome comparison of two Magnaporthe oryzae field isolates reveals genome variations and potential virulence effectors.

Cas9.

Uncoupling transcription from covalent histone modification.

Knockout of the adp gene related with colonization in Bacillus nematocida B16 using customized transcription activator-like effectors nucleases.

The genome-wide binding profile of the Sulfolobus solfataricus transcription factor Ss-LrpB shows binding events beyond direct transcription regulation.

Mechanogenetic regulation of transcription.

Transcription Regulation in Archaea.

Regulation of αENaC transcription.

Genome instability: Targeted telomere insertion.

Bunyavirus RNA synthesis: genome transcription and replication.